Journal: eLife
Article Title: Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism
doi: 10.7554/eLife.47198
Figure Lengend Snippet: ( a ) AMH released in the medium of COS-7 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Bar graph illustrates the mean amount of AMH secreted in the conditioned medium of transfected COS-7 cells ( n = 3 independent experiments per condition). Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test ( F 4,10 = 1193, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0001, AMH WT vs p.Thr99Ser p<0.0001). No significant motility difference was detected between Mock, p.Thr99Ser and p.Pro151Ser mutated forms of AMH treatment, p>0.9 for all. ( b ) Transwell assay was performed on GN11 cells transiently transfected either with lipofectamine alone (mock), or with a WT AMH or a variant AMH identified in CHH and KS probands. Comparisons between treatment groups were performed using a one-way ANOVA followed by Tukey’s post hoc comparison test ( F 4,50 = 13.94, Mock vs AMH WT p<0.0001, AMH WT vs p.Pro151Ser p<0.0001, AMH WT vs p.Asp238Glu p<0.0014, AMH WT vs p.Thr99Ser p = 0.0218. No significant motility difference was detected between Mock and mutated forms of AMH treatment, p>0.9 for all. ( c ) Quantification of GnRH secretion from GT1-7 cells transfected with lipofectamine alone (mock), or with a WT AMH or the p.Pro151Ser AMH variant identified in a nCHH proband. GnRH mean concentration measured in the medium ( n = 3, one-way ANOVA: F 2,6 = 43.84, p = 0.0003; followed by Tukey’s multiple comparison post hoc test, mock vs. AMH WT p = 0.0003, mock vs p.Thr99Ser p = 0.5220, AMH WT vs p.Thr99Ser p = 0.0007. ( d ) Transwell assay was performed on GN11 cells transiently transfected with the AMHR2 plasmid or with the AMHR2 variant and stimulated with either serum-free medium (SFM) or with recombinant AMH (50 ng/ml). Bar graph illustrates the mean number of migrated GN11 cells under different treatment conditions (SFM n = 10 for both WT and mutant AMHR2, AMH 50 ng/ml n = 12 for both WT and mutant AMHR2). Comparisons between treatment groups were performed using two-way ANOVA ( F 1,43 = 16.5 P = 0.0002; followed by Sidak’s multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml p<0.0001, p.Gly445_Leu453del SFM vs p.Gly445_Leu453del + AMH 50 ng/ml P = 0.1036). ( e ) Quantification of GnRH secretion from GT1-7 cells transfected with the same plasmids as in d (n = 3 independent experiments per condition). Experiments were replicated three times with comparable results. Two-way ANOVA, F 1,8 = 1.927, p<0.02025; followed by Holm-Šídák multiple comparison post hoc test, AMHR2 WT SFM vs AMHR2 WT + AMH 50 ng/ml P = 0.0269, p.Gly445_Leu453del SFM vs p.Gly445_Leu453del + AMH 50 ng/ml P = 0.4652. ( f ) Initial three-dimensional models of WT and p.Gly445_Leu453del catalytic intracellular serine/threonine domains of AMHR2. The backbone of the WT and deleted proteins are shown in tan or white cartoon representations, respectively, with the deleted 445–453 residues colored in red. The activation loop is depicted in blue. ( g–i ) Root-mean-square fluctuations (RMSF) of the C α atoms along the simulations for the AMHR2 WT and the p.Gly445_Leu453del models. ( g ) RMSF (in Å) for the WT (black line) and the p.Gly445_Leu453del models (red line, being the average over the three 100 ns simulations) are given for each residue of the protein. For a better comparison, residue numbers were kept the same for both models. Molecular representation of the WT ( h ) and p.Gly445_Leu453del ( i ) models colored by RMSF: the blue-green-red scale corresponds to low-medium-high RMSF values. The yellow spheres indicate the first residues after the p.Gly445_Leu453del deletion. The activation loop region is highlighted inside a blue frame (arrows). 10.7554/eLife.47198.019 Figure 7—source data 1. This spreadsheet contains the values used to generate the bar plots shown in .
Article Snippet: Commercial assay or kit , GnRH EIA kit , Phoenix Pharmaceuticals Inc , #EK-040-02CE , .
Techniques: Transfection, Variant Assay, Comparison, Transwell Assay, Concentration Assay, Plasmid Preparation, Recombinant, Mutagenesis, Activation Assay, Residue
